Cobalamin conjugates useful as antitumor agents

ABSTRACT

The invention provides cobalamin compounds linked to a neutron capture therapy target (e.g. Boron-10 or Gadolinium-157), and optionally linked to a detectable moiety, as well as pharmaceutical compositions comprising the compounds, and methods for using the compounds in medical diagnosis and therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/777,820, now allowed, filed on Feb. 12, 2004, which is a continuationof U.S. application Ser. No. 09/690,197, issued as U.S. Pat. No.6,806,363, filed Oct. 16, 2000, which claims priority to U.S.Provisional Application Ser. No. 60/129,733, filed Apr. 16, 1999; andU.S. Provisional Application Ser. No. 60/159,873, filed Oct. 15, 1999,all of which are herein incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

Boron neutron capture therapy is based on the nuclear reaction thatoccurs when a stable isotope, ¹⁰B, is irradiated with low energy (0.025eV) or thermal neutrons to yield helium nuclei (α-particles) and ⁷Linuclei.

The therapeutic potential of this reaction was recognized by Locher over50 years ago (Locher, G. L. et al., Am. J. Roentgenol. Radium Ther., 36,1-13 (1936)), but it was Sweet (Javid, M. et al., J. Clin. Invest., 31,603-610 (1952); Sweet, W. H., N. Engl. J. Med., 245, 875-878 (1951);Sweet, W. H. et al., J. Neurosurg., 9, 200-209 (1952)), who firstsuggested that boron neutron capture therapy (BNCT) might be useful forthe treatment of brain tumors.

Shortly thereafter, a clinical trial was initiated at the BrookhavenNational Laboratory in cooperation with Sweet and others at theMassachusetts General Hospital utilizing borax as the capture agent(Farr, L. E. et al., Am. J. Roentgenol., 71, 279-291 (1954); Godwin, J.T. et al., Cancer(Phila.), 8, 601-615 (1955)). The objective at thattime was to use BNCT as an adjunct to surgery for the treatment ofpatients with the most highly malignant and therapeutically refractoryof all brain tumors, glioblastoma multiforme.

Further trials were carried out in the early 1960s, but these failed toshow any evidence of therapeutic efficacy (Farr, L. E. et al., supra;Godwin, J. T. et al., supra; Asbury, A. K. et al., J. Neuropathol. Exp.Neurol., 31, 278-303 (1972)) and were associated with adverse effects innormal tissues (Asbury, A. K. et al., supra). Stimulated by the moreencouraging clinical studies of Hatanaka et al. (Hatanaka, H. A., J.Neurol., 209, 81-94 (1975); Hatanaka, H. et al., Boron Neutron CaptureTherapy for Tumors, Chap. 25, pp. 349-378. Niigata, Japan: NishimuraCo., Ltd. (1986)) for the treatment of malignant gliomas and those ofMishima et al. (Mishima, Y. et al, Lancet., 2, 388-289 (1989)) formelanoma, there has been renewed national and international interest inBNCT.

The theoretical advantage of BNCT is that it is a two component orbinary system, consisting of ¹⁰B and thermal neutrons, which whencombined together generate high linear energy transfer (LET) radiationcapable of selectively destroying tumor cells without significant damageto normal tissues. In order for BNCT to succeed a critical amount of ¹⁰Band a sufficient number of thermal neutrons must be delivered toindividual tumor cells.

Over the past few years the Department of Energy and the NIH haverenewed funding for BNCT-related research, and this has supported agrowing number of investigators in many different disciplines. Advancesin BNCT in the areas of compound distribution and pharmacokineticscompare favorably with other emerging modalities such as photonactivation therapy, photodynamic therapy, and the use of radiolabeledantibodies for cancer treatment in which physiological targeting isused.

There are a number of nuclides that have a high propensity for absorbinglow energy or thermal neutrons, and this property, referred to as theneutron capture cross-section (Σ), is measured in barns (1 b=10⁻²⁴ cm²).Of the various nuclides that have high neutron capture cross-sections,¹⁰B is the most attractive for the following reasons: (a) it isnonradioactive and readily available, comprising approximately 20% ofnaturally occurring boron: (b) the particles emitted by the capturereaction [¹⁰B(n,α)₇Li] are largely high LET: (c) their path lengths areapproximately 1 cell diameter (10-14 μm), theoretically limiting theradiation effect to those tumor cells that have taken up a sufficientamount of ¹⁰B and simultaneously sparing normal cells and (d) theextensive chemistry of boron is such that it can be incorporated into amultitude of different chemical structures.

⁷Li and α-particles are the primary fission product of the neutroncapture reaction with ¹⁰B α-Particles are relatively slow and give riseto closely spaced ionizing events that consist of tracks of sharplydefined columns. They have a path length of approximately 10 nm, arehigh LET, and destroy a wide variety of biologically active moleculesincluding DNA, RNA, and proteins. For these reasons there is little, ifany, cellular repair from α-particle-induced radiation injury.

Since the ¹⁰B(n,α)₇Li reaction will produce a significant biologicaleffect only when there is a sufficient fluence of thermal neutrons and acritical amount of ¹⁰B localized around, on, or within the cell, theradiation produced can be extremely localized thereby sparing normaltissue components. Thus, selectivity is simultaneously one of theadvantages and disadvantages of BNCT, since it requires delivery ofboron-10 to tumor cells in greater amounts than normal cells.

Ideally, boron compounds to be used for BNCT should have a highspecificity for malignant cells with concomitantly low concentrations inadjacent normal tissues and blood. Since it is desirable to confine theradiation solely to these cells, an intracellular and optimallyintranuclear localization of boron would be preferred.

Several boron-containing derivatives of chlorpromazine have beensynthesized (Nakagawa, T. et al., Chem. Pharm. Bull. (Tokyo), 24,778-781 (1976); Alam, F. et al., Sthralenther. Onkol., 165, 121-125(1989)) and are being evaluated for their in vivo tumor localizingproperties. p-Boronophenylalanine is another compound that is beingstudied as a potential capture agent for the treatment of melanoma. Therationale for its use is the avidity of melanomas for aromatic aminoacids and their subsequent incorporation into melanin (Ichihashi, M. etal., J. Invest. Dermatol., 78, 215-218 (1982); Mishima, Y. et al.,Neutral Capture Therapy, 230-236, Niigata, Japan: Nishimura Co., Ltd.(1986)).

Tumor localization has been demonstrated following I.V. administrationby means of whole body autoradiography (Coderre, J. A. et al., CancerRes., 48, 6313-6316 (1988)) and in several patients with cutaneousmelanoma following perilesional injection (Mishima, Y. et al,Sthralenther. Onkol., 165, 251-254 (1989)). Stimulated by Mishima'sexperience, a number of other boron-containing amino acids have beensynthesized that potentially could be incorporated in larger amountsinto proteins of malignant cells (Hall, I. H. et al., J. Pharm. Sci.,68, 685-688 (1979).

Another approach to the selective targeting of boron to melanomas isbased on the observation that thiouracil is preferentially incorporatedinto melanotic melanomas during melanogenesis (Whittacker, J. R., J.Biol. Chem., 246, 6217-6226 (1971)). This observation provided theimpetus for the synthesis of several boron-containing thiouracils(Gabel, D., Clinical Aspects of Neutron Capture Therapy, 233-241, NewYork: Plenum Publishing Co. (1989)), and these currently are beingevaluated in animals.

Two other classes of compounds with a propensity for localizing inmalignant tumors are the porphyrins and the related phthalocyanines. Thebiochemical basis by which these compounds achieve elevatedconcentration in malignant tumors is unknown, but this observation hasserved as the rationale for the use of hematoporphyrin derivative in thephotodynamic therapy of cancer (Dougherty, T. J. et al., PorphyrinPhotosensitization, 3-13, New York: Plenum Publishing Corp. (1981)).

The high concentration of these compounds in tumors and theirintracellular localization and persistence have stimulated severalgroups of investigators to synthesize boronated porphyrins (Kahl, S. B.et al., Neutron Capture Therapy, 61-67, Niigata, Japan: Nishimura Co.,Lid. (1986)) and phthalocyanines (Alam, F. et al., Strahlenther. Oncol.,165, 121-123 (1989)) as potential capture agents. Boronated porphyrinsappear to be 3-4 times more effective per unit dose in cell culture thanthe monomeric or dimeric form of Na₂B₁₂H₁₁SH (Laster, B. H. et al.,Strahlenther. Oncol., 165, 203-205 (1989)). Although liverconcentrations of these compounds are also high (Kahl, S. B. et al.,supra) this would not limit their use as a capture agent for thetreatment of brain tumors.

One final category-of low molecular weight boron compounds areboron-containing purines and pyrimidines and their nucleosides. Therationale for their development is that such compounds may beselectively incorporated into rapidly proliferating tumor cells andtrapped within the cell following their conversion to the correspondingnucleotide. Alternatively, these bases and their nucleosides mayfunction as analogues of naturally occurring precursors of nucleic acidsand become incorporated into nuclear DNA.

Cytoplasmic or preferably a nuclear localization of all of these boroncompounds would be advantageous since the heavy particles resulting fromthe capture reaction would deliver a greater proportion of their energyto intranuclear targets, thereby permitting lower boron concentrationsthan would have been required if the compounds were locatedextracellularly (Gabel, D. et al, Radiat. Res., 111, 14-25 (1987);Fairchild, R. G. et al, Int. J. Radiat. Oncol. Biol. Phys., 11, 831-840(1985)). Schinazi and Prusoff (Schinazi, R. F. et al., TetrahedronLett., 50, 4981-4984 (1978)) have synthesized the first boron-containingnucleoside, 5-dihydroloxyboryl-2′-deoxyuridine, an analogue ofthymidine, and have shown that it was not cytotoxic to African greenmonkey (i) cells at a concentration level of 1600 μM (Laster, B. H. etal., Neutron Capture Therapy, 46-54, Niigata, Japan: Nishimura Co., Ltd.(1986)). In vitro neutron radiation studies of cells grown in thepresence of 5-dihydroxy-2′-deoxyuridine produced a biological effectthat was equivalent to a concentration of 6 μg ¹⁰B/g, which, ifattainable in vivo, would be sufficient for BNCT.

During the 1960s and early 1970s, interests developed in the potentialuse of polyclonal antibodies directed against tumor-associated antigensfor the delivery of drugs and radioisotopes to tumors (Pressman, D. etal., Cancer Res., 40, 3001-3007 (1957); Ghose, T. et al., Br. Med. J.,1, 90-93 (1967); Ghose, T. et al., Cancer(Phila.), 29, 1398-1400(1972)). In 1964, Soloway suggested that antibodies might be used forthe selective targeting of ¹⁰B to tumors (Soloway, A. H., supra).Hawthorne et al. (Hawthorne, M. F. et al., J. Medicinal Chem., 15,449-452 (1972)) reported on the incorporation of the diazonium salt from1-(4-aminophenyl)-1,2-dicarbo-closo-dodecaborane into antibodiesdirected against bovine serum albumin and polyclonal antibodies directedagainst human and mouse histocompatability antigens (Hawthorne, M. F. etal., supra).

It was claimed from in vitro experiments that these immunoconjugateswere capable of delivering enough boron to human and murine lymphocytesto sustain a lethal ¹⁰B(n,α)⁷Li reaction, as evidenced by reducedviability following neutron irradiation. However, the immunoconjugatescontained only 0.2% natural boron by weight, which was equal to 6 atomsof ¹⁰B/molecule of antibody. In retrospect, it appears that there musthave been some other explanation for the reduced cell viability that wasobserved. Sneath et al (Sneath, R. L., Jr., J. Medicinal Chem., 17,796-799 (1974)) showed that water-solubilizing groups had to beincorporated into protein-binding polyhedral boranes if proteinsolubility in aqueous systems was to be maintained.

Subsequently, a group of polyhedral borane derivatives containingprotein-binding functional groups were linked to IgG molecules by meansof the carbodiimide reaction without evidence of precipitation (Sneath,R. L. et al., J. Medicinal Chem., 19, 1290-1294 (1976)).

One final category of macromolecular species that are possibly usefulfor the delivery of ¹⁰B is what may be termed “encapsulating complexes,”such as liposomes, microspheres, and low density lipoproteins (Kahl, S.B. et al, Strahlenther. Onkol., 165, 137-139 (1989)). Theoretically,large amounts of ¹⁰B could be encapsulated, and if these encapsulatingcomplexes could be targeted to the tumor by linkage to a monoclonalantibody using existing methodology or targeting an endogenouslyexpressed cell surface receptor, they might be powerful deliverysystems. Again, there may be preferential localization in thereticuloendothelial system, and methodology would have to be developedto minimize this and maximize tumor uptake.

Cobalamin

For several years after the isolation of vitamin B₁₂ as cyanocobalaminin 1948, it was assumed that cyanocobalamin and possiblyhydroxocobalamin, its photolytic breakdown product, occurred in man.Since then it has been recognized that cyanocobalamin is an artifact ofthe isolation of vitamin B₁₂ and that hydroxocobalamin and the twocoenzyme forms, methylcobalamin and adenosylcobalamin, are the naturallyoccurring forms of the vitamin.

The structure of these various forms is shown in FIG. 1, wherein X isCN, OH, CH₃ or adenosyl, respectively. Hereinafter, the term cobalaminwill be used to refer to all of the molecule except the X group. Thefundamental ring system without cobalt (Co) or side chains is calledcorrin and the octadehydrocorrin is called corrole. FIG. 1 is adaptedfrom The Merck Index, Merck & Co. (11th ed. 1989), wherein X is abovethe plane defined by the corrin ring and nucleotide is below the planeof the ring. The corrin ring has attached six amidoalkyl (H₂NC(O)Alk)substituents, at the 2, 3, 7, 8, 13, and 18 positions, which can bedesignated a-e and g, respectively. See D. L. Anton et al., J. Amer.Chem. Soc., 102, 2215 (1980).

Methylcobalamin serves as the cytoplasmic coenzyme for⁵N-methyltetrahydrofolate: homocysteine methyl transferase (methioninesynthase, EC 2.1.1.13), which catalyzes the formation of methionine fromhomocysteine. Adenosylcobalamin is the mitochondrial coenzyme formethylmalonyl CoA mutase (EC5.4.99.2) which interconverts methylmalonylCoA and succinyl CoA.

All forms of vitamin B₁₂ (adenosyl-, cyano-, hydroxo-, ormethylcobalamin) must be bound by the transport proteins, IntrinsicFactor and Transcobalamin II to be biologically active. Specifically,gastrointestinal absorption of vitamin B₁₂ relies upon the intrinsicfactor-vitamin B₁₂ complex being bound by the intrinsic factor receptorsin the terminal ileum. Likewise, intravascular transport and subsequentcellular uptake of vitamin B₁₂ throughout the body is dependent upontranscobalamin II and the cell membrane transcobalamin II receptors,respectively. After the transcobalamin II-vitamin B₁₂ complex has beeninternalized, the transport protein undergoes lysozymal degradation,which releases vitamin B₁₂ into the cytoplasm. All forms of vitamin B₁₂can then be interconverted into adenosyl-, hydroxo-, or methylcobalamindepending upon cellular demand. See, for example, A. E. Finkler et al.,Arch. Biochem. Biophys., 120, 79 (1967); C. Hall et al., J. CellPhysiol., 133, 187 (1987); M. E. Rappazzo et al., J. Clin. Invest., 51,1915 (1972) and R. Soda et al., Blood, 65, 795 (1985).

Cells undergoing rapid proliferation have been shown to have increaseduptake of thymidine and methionine. (See, for example, M. E. vanEijkeren et al., Acta Oncologica, 31, 539 (1992); K. Kobota et al., J.Nucl. Med., 32, 2118 (1991) and K. Higashi et al., J. Nucl. Med., 34,773 (1993)). Since methylcobalamin is directly involved with methioninesynthesis and indirectly involved in the synthesis of thymidylate andDNA, it is not surprising that methylcobalamin as well asCobalt-57-cyanocobalamin have also been shown to have increased uptakein rapidly dividing tissue (for example, see, B. A. Cooper et al.,Nature, 191, 393 (1961); H. Flodh, Acta Radiol. Suppl., 284, 55 (1968);L. Bloomquist et al., Experientia, 25, 294 (1969)). Additionally,up-regulation in the number of transcobalamin II receptors has beendemonstrated in several malignant cell lines during their acceleratedthymidine incorporation and DNA synthesis (see, J. Lindemans et al.,Exp. Cell. Res., 184 449 (1989); T. Amagasaki et al., Blood, 26, 138(1990) and J. A. Begly et al., J. Cell Physiol., 156, 43 (1993).

Vitamin B₁₂ has several characteristics which potentially make it anattractive in vivo tumor therapeutic agent. Vitamin B₁₂ is watersoluble, has no known toxicity, and in excess is excreted by glomerularfiltration. In addition, the uptake of vitamin B₁₂ could potentially bemanipulated by the administration of nitrous oxide and otherpharmacological agents (D. Swanson et al, Pharmaceuticals in MedicalImaging, MacMillan Pub. Co., NY (1990) at pages 621-628).

A process for preparing ¹²⁵I-vitamin B₁₂ derivatives is described inU.S. Pat. No. 3,981,863 issued to Niswender et al. In this process,vitamin B₁₂ is first subjected to mild hydrolysis to form a mixture ofmonocarboxylic acids, which Houts, infra, disclosed to contain mostlythe (e)-isomer. The mixture is then reacted with a p-(aminoalkyl)phenolto introduce a phenol group into the B₁₂ acids (via reaction with one ofthe free carboxylic acid groups). The mixed substituent B₁₂ derivativesare then iodinated in the phenol-group substituent. This U.S. patentteaches that the mixed ¹²⁵I-B₁₂ derivatives so made are useful in theradioimmunoassay of B₁₂, using antibodies raised against the mixture.

U.S. Pat. No. 4,465,775 issued to T. M. Houts reported that thecomponents of the radiolabeled mixture of Niswender et al. did not bindwith equal affinity to IF. Houts disclosed that radioiodinatedderivatives of the pure monocarboxylic (d)-isomer are useful in assaysof B₁₂ in which IF is used. However, although Houts generally disclosesthat the monocarboxylic (d)-isomer can be labeled with fluorophores orenzymes and used in competitive assays for vitamin B₁₂ in fluids, acontinuing need exists for labeled vitamin B₁₂ derivatives suitable fortumor and organ imaging and therapy.

U.S. Pat. No. 5,739,313 issued to Collins and Hogenkamp reported thatcobalamin analogs comprising a linking group and a chelating groupoptionally comprising a detectable radionuclide or a paramagnetic ionlocalize in tumor cells and are useful for imaging tumors.

Despite previous efforts to identify a neutron capture agent thatlocalizes in tumor cells in high concentration and is useful to treatcancer, there is currently a need for neutron capture agents that areuseful to treat tumors.

SUMMARY OF THE INVENTION

The invention provides a compound of the invention which is a residue ofa compound of formula I (FIG. 1) I linked to a residue of a moleculecomprising Boron-10 (i.e., B-10), wherein X is CN, OH, CH₃, adenosyl, ora molecule comprising B-10; or a pharmaceutically acceptable saltthereof.

The invention also provides a compound wherein a residue of a compoundof formula I (FIG. 1) is linked to a group of the formula Q-L-W-Det;wherein X is CN, OH, CH₃, adenosyl, a molecule comprising B-10, orQ-L-W-Det; wherein Det is a chelating group comprising Gd-157; L is alinker or absent; and W and Q are each independently —N(R)C(═O)—,—C(═O)N(R)—, —OC(═O)—, —C(═O)O—, —O—, —S—, —S(O)—, —S(O)₂—, —C(═O)—,—N(R)—, or a direct bond; wherein each R is independently H or(C₁-C₆)alkyl; or a pharmaceutically acceptable salt thereof.

The invention also provides a compound wherein a residue of a compoundof formula I (FIG. 1) is linked to a residue of a molecule comprisingB-10; wherein a residue of the compound of formula I is also linked to agroup of the formula Q-L-W-Det, wherein X is CN, OH, CH₃, adenosyl, agroup of the formula Q-L-W-Det, or a molecule comprising B-10; wherein:Det is a chelating group comprising a therapeutic radionuclide or adiagnostic radionuclide; L is a linker or absent; and Q and W are eachindependently —N(R)C(═O)—, —C(═O)N(R)—, —OC(═O)—, —C(═O)O—, —O—, —S—,—S(O)—, —S(O)₂—, —C(═O)—, —N(R)—, or a direct bond; wherein each R isindependently H or (C₁-C₆)alkyl; or a pharmaceutically acceptable saltthereof.

The invention also provides a compound wherein a residue of a compoundof formula I (FIG. 1) is linked to a residue of a molecule comprisingB-10; wherein the residue of the compound of formula I is also linked toa group of the formula Q-L-W-Det, wherein X is CN, OH, CH₃, adenosyl, agroup of the formula Q-L-W-Det, or a molecule comprising B-10; Det is achelating group comprising Gd-157; L is a linker or absent; and Q and Ware each independently —N(R)C(═O)—, —C(═O)N(R)—, —OC(═O)—, —C(—O)O—,—O—, —S—, —S(O)—, —S(O)₂—, —C(═O)—, —N(R)—, or a direct bond; whereineach R is independently H or (C₁-C₆)alkyl; or a pharmaceuticallyacceptable salt thereof.

The invention also provides a compound wherein a residue of a compoundof formula I (FIG. 1) is linked to a detectable radionuclide and alsolinked to a residue of a molecule comprising Boron-10 (i.e., B-10),wherein X is CN, OH, CH₃, adenosyl, or a molecule comprising B-10; or apharmaceutically acceptable salt thereof.

The invention also provides a compound wherein a residue of a compoundof formula I (FIG. 1) is linked to a detectable radionuclide; and isalso linked to a group comprising Gd-157; wherein X is CN, OH, CH₃,adenosyl, a molecule comprising B-10, or a group comprising Gd-157; or apharmaceutically acceptable salt thereof. Specifically, the groupcomprising Gd-157 can have the formula Q-L-W-Det; wherein Det is achelating group comprising Gd-157; L is a linker or absent; and W and Qare each independently —N(R)C(═O)—, —C(═O)N(R)—, —OC(═O)—, —C(═O)O—,—O—, —S—, —S(O)—, —S(O)₂—, —C(═O)—, —N(R)—, or a direct bond; whereineach R is independently H or (C₁-C₆)alkyl.

The invention also provides a compound of wherein a residue of acompound of formula I (FIG. 1) is linked 1) to a molecule comprisingB-10 or to a chelating group comprising Gd-157, and is linked 2) to atleast one residue of the formula Q-L-W-Det; wherein each Det isindependently a chelating group comprising a metallic radionuclide; eachL is independently a linker or absent; and each W and Q is independently—N(R)C(═O)—, —C(═O)N(R)—, —OC(═O)—, —C((═O)O—, —O—, —S—, —S(O)—,—S(O)₂—, —C(═O)—, —N(R)—, or a direct bond; wherein each R isindependently H or (C₁-C₆)alkyl; or a pharmaceutically acceptable saltthereof.

The invention also provides a pharmaceutical composition comprising acompound of the present invention and a pharmaceutically acceptablecarrier.

The invention also provides a method of treating a tumor in a mammal inneed of such treatment comprising administering to the mammal aneffective amount of a compound of the present invention; andadministering neutron capture therapy.

The invention also provides a method for imaging a tumor in a mammal inneed of such imaging comprising administering to the mammal a detectableamount of a compound of the present invention in combination with apharmaceutically acceptable vehicle effective to image the tumor; anddetecting the presence of the compound.

The invention also provides a compound of the present invention for usein medical therapy or diagnosis.

The invention also provides the use of a compound of the presentinvention for the manufacture of a medicament for imaging a tumor in amammal (e.g., a human).

The invention also provides the use of a compound of the presentinvention for the manufacture of a medicament for treating a tumor in amammal (e.g., a human).

The invention also provides intermediates disclosed herein that areuseful in the preparation of the compounds of the present invention aswell as synthetic methods useful for preparing the compounds of theinvention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the structure of cobalamin wherein X is CN (cyano), OH,CH₃ or adenosyl.

FIG. 2 illustrates the synthesis of a cyanocobalamin-nido-carboraneconjugate.

FIG. 3 illustrates the synthesis of representative compounds of thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION

Applicant has discovered certain cobalamin analogs are useful, incombination with neutron capture therapy, to treat tumors. Applicant hasalso discovered certain cobalamin analogs are useful as neutron captureagents and as tumor imaging agents.

The following definitions are used, unless otherwise described: halo isfluoro, chloro, bromo, or iodo. Alkyl, alkoxy, alkenyl, alkynyl, etc.denote both straight and branched groups; but reference to an individualradical such as “propyl” embraces only the straight chain radical, abranched chain isomer such as “isopropyl” being specifically referredto. Aryl denotes a phenyl radical or an ortho-fused bicyclic carbocyclicradical having about nine to ten ring atoms in which at least one ringis aromatic.

Specific and preferred values listed below for radicals, substituents,and ranges, are for illustration only; they do not exclude other definedvalues or other values within defined ranges for the radicals andsubstituents.

It is appreciated that those skilled in the art will recognize thatcompounds of the present invention having a chiral center may exist inand be isolated in optically active and racemic forms. Some compoundsmay exhibit polymorphism. It is to be understood that the presentinvention encompasses any racemic, optically-active, polymorphic, orstereoisomeric form, or mixtures thereof, of a compound of theinvention, which possess the useful properties described herein, itbeing well known in the art how to prepare optically active forms (forexample, by resolution of the racemic form by recrystallizationtechniques, by synthesis from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase) and how to determine antitumor activity or utility asan antitumor imaging agent using the standard tests described herein, orusing other similar tests which are well known in the art.

Specifically, (C₁-C₁₄)alkyl can be methyl, ethyl, propyl, isopropyl,butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, hexyl, heptyl, octyl,nonyl, decyl, undecyl, dodecyl, tridecyl or tetradecyl.

Specifically, (C₂-C₁₄)alkenyl can be vinyl, allyl, 1-propenyl,2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl,3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl,5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl,6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl,6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl,5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl,3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl,9-decenyl, 1-undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl,5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl,10-undecenyl, 1-dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl,5-dodecenyl, 6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl,10-dodecenyl, 11-dodecenyl, 1-tridecenyl, 2-tridecenyl, 3-tridecenyl,4-tridecenyl, 5-tridecenyl, 6-tridecenyl, 7-tridecenyl, 8-tridecenyl,9-tridecenyl, 10-tridecenyl, 11-tridecenyl, 12-tridecenyl,1-tetradecenyl, 2-tetradecenyl, 3-tetradecenyl, 4-tetradecenyl,5-tetradecenyl, 6-tetradecenyl, 7-tetradecenyl, 8-tetradecenyl,9-tetradecenyl, 10-tetradecenyl, 11-tetradecenyl, 12-tetradecenyl or13-tetradecenyl.

Specifically, (C₂-C₁₄)alkynyl can be ethynyl, 1-propynyl, 2-propynyl,1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl,4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl,1-heptynyl, 2-heptynyl, 3-heptynyl, 4-heptynyl, 5-heptynyl, 6-heptynyl,1-octynyl, 2-octynyl, 3-octynyl, 4-octynyl, 5-octynyl, 6-octynyl,7-octynyl, 1-nonylyl, 2-nonynyl, 3-nonynyl, 4-nonynyl, 5-nonynyl,6-nonynyl, 7-nonynyl, 8-nonynyl, 1-decynyl, 2-decynyl, 3-decynyl,4-decynyl, 5-decynyl, 6-decynyl, 7-decynyl, 8-decynyl, 9-decynyl,1-undecynyl, 2-undecynyl, 3-undecynyl, 4-undecynyl, 5-undecynyl,6-undecynyl, 7-undecynyl, 8-undecynyl, 9-undecynyl, 10-undecynyl,1-dodecynyl, 2-dodecynyl, 3-dodecynyl, 4-dodecynyl, 5-dodecynyl,6-dodecynyl, 7-dodecynyl, 8-dodecynyl, 9-dodecynyl, 10-dodecynyl,11-dodecynyl, 1-tridecynyl, 2-tridecynyl, 3-tridecynyl, 4-tridecynyl,5-tridecynyl, 6-tridecynyl, 7-tridecynyl, 8-tridecynyl, 9-tridecynyl,10-tridecynyl, 11-tridecynyl, 12-tridecynyl, 1-tetradecynyl,2-tetradecynyl, 3-tetradecynyl, 4-tetradecynyl, 5-tetradecynyl,6-tetradecynyl, 7-tetradecynyl, 8-tetradecynyl, 9-tetradecynyl,10-tetradecynyl, 11-tetradecynyl, 12-tetradecynyl or 13-tetradecynyl.

Specifically “aryl” can be phenyl, indenyl, or naphthyl.

Specifically (C₃-C₈)cycloalkyl can be cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl.

As used herein, an “amino acid” is a natural amino acid residue (e.g.Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Hyl, Hyp, Ile, Leu, Lys,Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D or L form, as well asunnatural amino acid (e.g. phosphoserine; phosphothreonine;phosphotyrosine; hydroxyproline; gamma-carboxyglutamate; hippuric acid;octahydroindole-2-carboxylic acid; statine;1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid; penicillamine;ornithine; citruline; α-methyl-alanine; para-benzoylphenylalanine;phenylglycine; propargylglycine; sarcosine; and tert-butylglycine)residue having one or more open valences. The term also comprisesnatural and unnatural amino acids bearing conventional amino protectinggroups (e.g. acetyl, acyl, trifluoroacetyl, or benzyloxycarbonyl), aswell as natural and unnatural amino acids protected at carboxy (e.g. asa (C₁-C₆)alkyl, phenyl or benzyl ester or amide) with conventionalprotecting groups. Other suitable amino and carboxy protecting groupsare known to those skilled in the art (See for example, T. W. Greene,Protecting Groups In Organic Synthesis; Wiley: New York, 1981; D. Voet,Biochemistry, Wiley: New York, 1990; L. Stryer, Biochemistry, (3rd Ed.),W. H. Freeman and Co.: New York, 1975; J. March, Advanced OrganicChemistry, Reactions, Mechanisms and Structure, (2nd Ed.), McGraw Hill:New York, 1977; F. Carey and R. Sundberg, Advanced Organic Chemistry,Part B: Reactions and Synthesis, (2nd Ed.), Plenum: New York, 1977; andreferences cited therein). According to the invention, the amino orcarboxy protecting group can also comprise a radionuclide (e.g.,Fluorine-18, Iodine-123, or Iodine-124).

As used herein, a “peptide” is a sequence of 2 to 25 amino acids (e.g.as defined hereinabove) or peptidic residues having one or more openvalences. The sequence may be linear or cyclic. For example, a cyclicpeptide can be prepared or may result from the formation of disulfidebridges between two cysteine residues in a sequence. A peptide can belinked through the carboxy terminus, the amino terminus, or through anyother convenient point of attachment, such as, for example, through thesulfur of a cysteine. Peptide derivatives can be prepared as disclosedin U.S. Pat. Nos. 4,612,302; 4,853,371; and 4,684,620, or as describedin the Examples herein below. Peptide sequences specifically recitedherein are written with the amino terminus on the left and the carboxyterminus on the right.

Particular and specific values listed below for radicals, substituents,and ranges, are for illustration only and they do not exclude otherdefined values or other values within defined ranges for the radicalsand substituents.

Specifically, the peptide can be poly-L-lysine, poly-L-glutamic acid,poly-L-aspartic acid, poly-L-histidine, poly-L-ornithine, poly-L-serine,poly-L-threonine, poly-L-tyrosine, poly-L-lysine-L-phenyl-alanine orpoly-L-lysine-L-tyrosine.

As used herein, a “residue of a compound of formula I” is a radical of acompound of formula I having one or more open valences. Anysynthetically feasible atom or atoms of the compound of formula I may beremoved to provide the open valence, provided the resulting compound isable to localize in or near tumors. Based on the linkage that isdesired, one skilled in the art can select suitably functionalizedstarting materials that can be derived from a compound of formula Iusing procedures that are known in the art. For example, suitable atomsthat may be removed include the NH₂ group of the a-carboxamide(illustrated in FIG. 1) or a hydrogen atom from the NH₂ group of thea-carboxamide, the NH₂ group of the b-carboxamide (illustrated inFIG. 1) or a hydrogen atom from the NH₂ group of the b-carboxamide, theNH₂ group of the d-carboxamide (illustrated in FIG. 1) or a hydrogenatom from the NH₂ group of the d-carboxamide, the NH₂ group of thee-carboxamide (illustrated in FIG. 1) or a hydrogen atom from the NH₂group of the e-carboxamide, and X at the 6-position (illustrated in FIG.1). In addition, the hydrogen atom of the hydroxy group at the 3′position of the sugar, the hydrogen atom from the hydroxyl group at the3′ position of the sugar, the hydrogen atom of the CH₂OH group at the 5′position, or the hydrogen atom from the hydroxyl group at the 5′position of the sugar ring may be removed.

As used herein, “adenosyl” is an adenosine radical in which anysynthetically feasible atom or group of atoms have been removed, therebyproviding an open valence.

Synthetically feasible atoms which may be removed include the hydrogenatom of the hydroxy group at the 5′ position. Accordingly, adenosyl canconveniently be attached to the 6-position of a compound of formula Ivia the 5′ position of adenosyl.

As used herein, a “molecule comprising B-10” can be any compound thatcontains at least one B-10 atom. The nature of the molecule thatincludes B-10 is not critical. The compound, however, must be nontoxicand must be able to enter the tumor cell or locate near the tumor cellwhen the molecule comprising B-10 is attached.

As used herein, a “residue of a molecule comprising B-10” is a radicalof a molecule comprising B-10 having one or more open valences. Anysynthetically feasible atom or atoms of the molecule comprising B-10 maybe removed to provide the open valence, provided bioactivity issubstantially retained. Based on the linkage that is desired, oneskilled in the art can select suitably functionalized starting materialsthat can be derived from a molecule comprising B-10 using proceduresthat are known in the art.

As used herein, a “residue of o-carborane,” a “residue of m-carborane,”or a “residue of p-carborane” is a radical of o-carborane, m-carboraneor p-carborane, respectively, having one or more open valences. Anysynthetically feasible atom or atoms of o-carborane, m-carborane orp-carborane may be removed to provide the open valence, providedbioactivity is substantially retained. Based on the linkage that isdesired, one skilled in the art can select suitably functionalizedstarting materials that can be derived from o-carborane, m-carborane orp-carborane using procedures that are known in the art.

Boron Compounds Useful for Neutron Capture Therapy

The invention provides a compound of formula I (FIG. 1) linked to one ormore molecules comprising B-10, wherein X is CN, OH, CH₃, adenosyl or amolecule comprising B-10; or a pharmaceutically acceptable salt thereof.

A variety of molecules comprising B-10 known in the art are useful inthe present invention. The molecules vary considerably in structure butare suitable, to practice the present invention. Acceptable speciesinclude boron containing amino acids, carbohydrates, and nucleosides, aswell as carboranes. A wide variety of boron-containing compounds arecommercially available, or are known in the art. A variety of moleculescomprising B-10 are commercially available from Boron Biologicals, Inc.,Raleigh, N.C. and RysCor Science, Inc., Raleigh, N.C.

Specifically, at least one molecule comprising B-10 can be o-carborane,m-carborane or p-carborane. More specifically, at least one moleculecomprising B-10 is o-carborane. o-Carborane[1,2-dicarbadodecaborane(12)]; m-carborane[1,7-dicarbadodecaborane(12)];and p-carborane[1,12-dicarbadodecaborane(12)] are commercially availablefrom Aldrich, Milwaukee, Wis.

Specifically, each molecule comprising B-10 can independently beo-carborane, m-carborane or p-carborane. More specifically, eachmolecule comprising B-10 is o-carborane.

Compound of Formula I/Molecule Comprising B-10

The residue of molecule comprising B-10 can be linked to the residue ofa compound of formula I through an amide (e.g., —N(R)C(═O)— or—C(═O)N(R)—), ester (e.g., —OC(═O)— or —C(═O)O—), ether (e.g., —O—),amino (e.g., —N(R)—), ketone (e.g., —C(═O)—), thioether (e.g., —S—),sulfinyl (e.g., —S(O)—), sulfonyl (e.g., —S(O)₂—), or a direct (e.g.,C—C bond) linkage, wherein each R is independently H or (C₁-C₆)alkyl.Such a linkage can be formed from suitably functionalized startingmaterials using synthetic procedures that are known in the art. Based onthe linkage that is desired, one skilled in the art can select suitablyfunctional starting materials that can be derived from a residue of acompound of formula I and from a given residue of a molecule comprisingB-10 using procedures that are known in the art.

The residue of the molecule comprising B-10 can be directly linked toany synthetically feasible position on the residue of a compound offormula I. Suitable points of attachment include, for example, theb-carboxamide, the d-carboxamide, and the e-carboxamide (illustrated inFIG. 1), as well as the 6-position (the position occupied by X in FIG.1), and the 5′-hydroxy and the 3′-hydroxy groups on the 5-membered sugarring, although other points of attachment are possible. U.S. Pat. No.5,739,313 discloses compounds (e.g.,cyanocobalamin-b-(4-aminobutyl)amide-,methylcobalamin-b-(4-aminobutyl)amide, andadenosylcobalamin-b-(4-aminobutyl)amide) that are useful intermediatesfor the preparation of compounds of the present invention.

Compounds wherein the residue of a molecule comprising B-10 is linked tothe 6-position of a compound of formula I can be prepared by reducing acorresponding Co (II) compound of formula I to form a nucleophilic Co(I) compound and treating this Co (I) compound with a residue of amolecule comprising B-10 (or a derivative thereof) comprising a suitableleaving group, such as a halide (e.g., a chloride).

The invention also provides compounds having more than one residue of amolecule comprising B-10 directly linked to a compound of formula I. Forexample, the residue of a molecule comprising B-10 can be directlylinked to a residue of the b-carboxamide of the compound of formula Iand a residue of another molecule comprising B-10 can be directly linkedto a residue of the d-carboxamide of the compound of formula I. Inaddition, the residue of a molecule comprising B-10 can be directlylinked to the 6-position of the compound of formula I and a residue ofanother molecule comprising B-10 can be directly linked to a residue ofthe d- or e-carboxamide of the compound of formula I.

Compound of Formula I/Linker/Molecule Comprising B-10 Linker

In addition to being directly linked to the residue of a compound offormula I, the residue of a molecule comprising B-10 can also be linkedto the residue of a compound of formula I by a suitable linker. Thestructure of the linker is not crucial, provided it yields a compound ofthe invention which has an effective therapeutic index against thetarget cells, and which will localize in or near tumor molecules.

Suitable linkers include linkers that separate the residue of a compoundof formula I and the residue of a molecule comprising B-10 by about 5angstroms to about 200 angstroms. Other suitable linkers include linkersthat separate the residue of a compound of formula I and the residue ofa molecule comprising B-10 by about 5 angstroms to about 100 angstroms,as well as linkers that separate the residue of a compound of formula Iand the residue of a molecule comprising B-10 by about 5 angstroms toabout 50 angstroms, or by about 5 angstroms to about 25 angstroms.Suitable linkers are disclosed, for example, in U.S. Pat. No. 5,735,313.

The linker can be linked to any synthetically feasible position on theresidue of a compound of the residue of formula I. Suitable points ofattachment include, for example, a residue of the b-carboxamide, aresidue of the d-carboxamide, and a residue of the e-carboxamide, the6-position (i.e., the position occupied by X in the compound of formulaI), as well as a residue of the 5′-hydroxy group and a residue of the3′-hydroxy group on the 5-membered sugar ring, although other points ofattachment are possible. Based on the linkage that is desired, oneskilled in the art can select suitably functionalized starting materialsthat can be derived from a compound of formula I and from a givenmolecule comprising B-10 using procedures that are known in the art.

The linker can conveniently be linked to the residue of a compound offormula I or to the residue of a molecule comprising B-10 through anamide (e.g., —N(R)C(O)— or —C(═O)N(R)—), ester (e.g., —OC(═O)— or—C(═O)O—), ether (e.g., —O—), ketone (e.g., —C(═O)—) thioether (e.g.,—S—), sulfinyl (e.g., —S(O)—), sulfonyl (e.g., —S(O)₂—), amino (e.g.,—N(R)—) or a direct (e.g., C—C) linkage, wherein each R is independentlyH or (C₁-C₆)alkyl. The linkage can be formed from suitablyfunctionalized starting materials using synthetic procedures that areknown in the art. Based on the linkage that is desired, one skilled inthe art can select suitably functional starting materials that can bederived from a residue of a compound of formula I, a residue of amolecule comprising B-10, and from a given linker using procedures thatare known in the art.

Specifically, the linker can be a divalent radical of the formula W-A-Qwherein A is (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl,(C₃-C₈)cycloalkyl, or (C₆-C₁₀)aryl, wherein W and Q are eachindependently —N(R)C(═O)—, —C(═O)N(R)—, —OC(═O)—, —C(═O)O—, —O—, —S—,—S(O)—, —S(O)₂—, —N(R)—, —C(═O)—, or a direct bond (i.e., W or Q isabsent); wherein each R is independently H or (C₁-C₆)alkyl.

Specifically, the linker can be a divalent radical of the formulaW—(CH₂)_(n)-Q wherein, n is between about 1 and about 20, between about1 and about 15, between about 2 and about 10, between about 2 and about6, or between about 4 and about 6; wherein W and Q are eachindependently —N(R)C(═O)—, —C(═O)N(R)—, —OC(═O)—, —C(═O)O—, —O—, —S—,—S(O)—, —S(O)₂—, —C(═O)—, —N(R)—, or a direct bond (i.e., W or Q isabsent); wherein each R is independently H or (C₁-C₆)alkyl.

Specifically, W and Q are each independently —N(R)C(═O)—, —C(═O)N(R)—,—OC(═O)—, —N(R)—, —C(═O)O—, —O—, or a direct bond (i.e., W or Q isabsent).

Specifically, the linker is a divalent radical, i.e., 1,ω-divalentradicals formed from a peptide or an amino acid. The peptide cancomprise 2 to about 20 amino acids, 2 to about 15 amino acids, or 2 toabout 12 amino acids.

Specifically, the peptide can be poly-L-lysine (i.e.,[—NHCH[(CH₂)₄NH₂]CO—]_(m)-Q, wherein Q is H, (C₁-C₁₄)alkyl, or asuitable carboxy protecting group; and wherein m is about 2 to about 20.Specifically, the poly-L-lysine can contains about 5 to about 15residues (i.e., m is between about 5 and about 15). More specifically,the poly-L-lysine can contain about 8 to about 11 residues (i.e., m isbetween about 8 and about 11).

Specifically, the peptide can be poly-L-glutamic acid, poly-L-asparticacid, poly-L-histidine, poly-L-ornithine, poly-L-serine,poly-L-threonine, poly-L-tyrosine, poly-L-lysine-L-phenylalanine orpoly-L-lysine-L-tyrosine.

Specifically, the linker is prepared from 1,6-diaminohexaneH₂N(CH₂)₆NH₂, 1,5-diaminopentane H₂N(CH₂)₁₄NH₂, 1,4-diaminobutaneH₂N(CH₂)₄NH₂, or 1,3-diaminopropane H₂N(CH₂)₃NH₂.

The linker can comprise one or more non-metallic radionuclides.Specifically, the linker can comprise more than one non-metallicradionuclides. More specifically, the linker can comprise 2 to about 10,2 to about 8, 2 to about 6, or 2 to about 4 non-metallic radioisotopes.

A specific residue of a peptide (i.e., linker) comprising one or morenon-metallic radionuclides has the following formula 1

wherein each M is independently a non-metallic radionuclide; each R isindependently (C₁-C₁₄)alkyl, (C₂-C₁₄)alkenyl, (C₂-C₁₄)alkynyl,(C₁-C₁₄)alkoxy, hydroxy, cyano, nitro, halo, trifluoromethyl,N(R_(a))(R_(b)), (C₁-C₁₄)alkanoyl, (C₂-C₁₄)alkanoyloxy, (C₆-C₁₀)aryl, or(C₃-C₈)cycloalkyl wherein R_(a) and R_(b) are each independently H or(C₁-C₁₄)alkyl; P; Q is H, (C₁-C₁₄)alkyl, or a suitable carboxyprotecting group; n is 2 to about 20; I is 1-5, j is 0-4 and I+j is <5;or a pharmaceutically acceptable salt thereof.

Specifically, i can be 1, j can be 0, M can be Fluorine-18, Bromine-76,or Iodine-123, and n can be about 6 to about 12.

The molecule comprising B-10 can comprise one or more boron atoms. Asuitable molecule comprising B-10 can contain 1 to about 20, 1 to about15, 1 to about 10, or 1 to about 5 boron atoms. In addition, themolecule comprising B-10 can comprise one or more B-10 atoms. A suitablemolecule comprising B-10 can contain 1 to about 20, 1 to about 15, 1 toabout 10, or 1 to about 5 B-10 atoms.

The molecule comprising B-10 can be an amino acid, a carbohydrate, anucleoside or a carborane. Specifically, the molecule comprising B-10 iso-carborane, m-carborane or p-carborane.

Compounds wherein the linker is linked to the 6-position of a compoundof formula I can be prepared by preparing a nucleophilic Co (I) speciesas described herein above, and reacting it with a linker comprising asuitable leaving group, such as a halide (e.g. a chloride).

The invention also provides compounds having more than one moleculecomprising B-10 attached to a compound of formula I, each through alinker. For example, the residue of a molecule comprising B-10 canconveniently be linked, through a linker, to a residue of theb-carboxamide of the compound of formula I and a residue of anothermolecule comprising B-10 can conveniently be linked, through a linker,to a residue of the d- or e-carboxamide of the compound of formula I. Inaddition, the residue of a molecule comprising B-10 can conveniently belinked, through a linker, to the 6-position of the compound of formula Iand a residue of another molecule comprising B-10 can conveniently belinked, through a linker, to a residue of the b-, d- or e-carboxamide ofthe compound of formula I.

The invention also provides compounds having more than one moleculecomprising B-10 attached to a compound of formula I, either directly orthrough a linker. For example, the residue of a molecule comprising B-10can conveniently be linked, either directly or through a linker, to aresidue of the b-carboxamide of the compound of formula I and a residueof another molecule comprising B-11 can conveniently be linked, eitherdirectly or through a linker, to a residue of the d- or e-carboxamide ofthe compound of formula I. In addition, the residue of a moleculecomprising B-10 can conveniently be linked, either directly or through alinker, to the 6-position of the compound of formula I and a residue ofanother molecule comprising B-10 can conveniently be linked, eitherdirectly or through a linker, to a residue of the b-, d- ore-carboxamide of the compound of formula I.

Compound of Formula I/Chelating Group Comprising Gadolinium-157

U.S. Pat. No. 5,739,313 discloses cobalamin analogs comprising acompound of formula I, a linking group, and a chelating group comprisinga detectable radionuclide or a paramagnetic ion. The compounds aredisclosed to localize. in tumor cells following administration, and tobe useful for imaging tumors.

The metallic radionuclide Gadolinium-157 is an especially useful ion forconducting magnetic resonance imaging. It is also a useful target ionfor neutron capture therapy. Applicants have discovered thatincorporation of Gd-157 into one of the chelating molecules disclosed inU.S. Pat. No. 5,739,313, provides a compound that is not onlyparticularly useful for conducting magnetic resonance imaging, but alsoa compound that can be used in conjunction with neutron capture therapy,to treat tumors.

Thus, the present invention provides a residue of a compound of formulaI linked to at least one residue of the formula -Q-L-W-Det; wherein eachDet is independently a chelating group comprising Gd-157; each L isindependently linker (as defined hereinabove) or is absent; and each Wand Q are each independently —N(R)C(═O)—, —C(═O)N(R)—, —OC(═O)—,—C(═O)O—, —O—, —S—, —S(O)—, —S(O)₂—, —C(═O)—, —N(R)—, or a direct bond(i.e., W or Q is absent); wherein each R is independently H or(C₁-C₆)alkyl.

Compound of Formula I/Chelating Group Comprising a Radionuclide

Applicant has also discovered that it is possible to prepare a compoundthat is useful for both imaging and for treating tumors by incorporatingone or more neutron capture target atoms (e.g. B-10 or Gd-157) into acompound that also comprises a detectable radionuclide. Accordingly, theinvention provides a residue of a compound of formula I which is linkedto one or more residues of a molecule comprising B-10; and which is alsolinked to one or more groups of the formula -Q-L-W-Det; wherein each Detis independently a chelating group (as defined herein) comprisingGd-157; each L is independently linker (as defined herein) or is absent;and each W and Q are each independently —N(R)C(═O)—, —C(═O)N(R)—,—OC(═O)—, —C(═O)O—, —O—, —S—, —S(O)—, —S(O)₂—, —C(═O)—, —N(R)—, or adirect bond (i.e., W or Q is absent); wherein each R is independently Hor (C₁-C₆)alkyl; or a pharmaceutically acceptable salt thereof.

The present invention provides a residue of a compound of formula Ilinked to a molecule comprising B-10 or linked to a chelating groupcomprising Gd-157 and wherein the residue of a compound of formula I islinked to at least one residue of the formula -Q-L-W-Det; wherein eachDet is independently a chelating group (as defined hereinabove)comprising a metallic radionuclide; each L is independently a linker (asdefined hereinabove) or is absent; and each W and Q are eachindependently N(R)C(═O)—, —C(═O)N(R)—, —OC(═O)—, —C(═O)O—, —O—, —S—,—S(O)—, —S(O)₂—, —C(═O)—, —N(R)—, or a direct bond (i.e., W or Q isabsent); wherein each R is independently H or (C₁-C₆)alkyl.

Compound of Formula I/Molecule Comprising B-110/Detectable Radionuclide

The invention also provides a compound wherein a residue of a compoundof formula I (FIG. 1) is linked 1) to a residue of a molecule comprisingB-10, and 2) to a detectable radionuclide, wherein X is CN, OH, CH₃,adenosyl, or a molecule comprising B-10; or a pharmaceuticallyacceptable salt thereof.

The detectable radionuclide can be directly linked to the residue of acompound of formula I, or can be linked by a linker to the residue of acompound of formula I. The linker can be any suitable linker describedherein. Specifically, the linker can be of the formula W-A wherein A is(C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₃-C₆)cycloalkyl, or(C₆-C₁₀)-aryl, wherein W is —N(R)C(═O)—, —C(═O)N(R)—, —OC(═O)—,—C(═O)O—, —O—, —S—, —S(O)—, —S(O)₂—, —N(R)—, —C(═O)—, or a direct bond;wherein each R is independently H or (C₁-C₆)alkyl; and wherein A issubstituted with one or more non-metallic radionuclides. The linker canalso be a peptide or an amino acid.

Compound of Formula I/Group Comprising Gd-157/Detectable Radionuclide

The invention also provides a compound wherein a residue of a compoundof formula I (FIG. 1) is linked 1) to a detectable radionuclide; and 2)to a group comprising Gd-157; or a pharmaceutically acceptable saltthereof.

The Gd-157 can be linked to the residue of a compound of formula I byany suitable means. For example, the residue of a compound of formula Ican be attached to a group comprising Gd-157 that has the formulaQ-L-W-Det, wherein Det is a chelating group comprising Gd-157, L is alinker or absent, and W and Q are each independently —N(R)C(═O)—,—C(═O)N(R) —, —OC(═O)—, —C(═O)O—, —O—, —S—, —S(O)—, —S(O)₂—, —C(═O)—,—N(R)—, or a direct bond; wherein each R is independently H or(C₁-C₆)alkyl. Any suitable chelator can be used.

The detectable radionuclide can be directly linked to the residue of acompound of formula I, or can be linked by a linker to the residue of acompound of formula I. The linker can be any suitable linker describedherein. Specifically, the linker can be is of the formula W-A wherein Ais (C₁-C₆)alkyl, (C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₃-C₈)cycloalkyl, or(C₆-C₁₀)aryl, wherein W is —N(R)C(═O)—, —C(═O)N(R)—, —OC(═O)—, —C(═O)O—,—O—, —S—, —S(O)—, —S(O)₂—, —N(R)—, —C(═O)—, or a direct bond; whereineach R is independently H or (C₁-C₆)-alkyl; and wherein A is substitutedwith one or more non-metallic radionuclides. The linker can also be apeptide or an amino acid

Non-Metallic Radionuclide

Any detectable non-metallic radionuclide that is suitable for imagingcan be used in the compounds of the invention. For example, suitablenon-metallic radionuclides include Carbon-11, Fluorine-18, Bromine-76,and Iodine-123. Specifically, the non-metallic radionuclide can be anon-metallic paramagnetic atom (e.g., Fluorine-19); or a non-metallicpositron emitting radionuclide (e.g., Carbon-1, Fluorine-18, Iodine-123,or Bromine-76).

Metallic Radionuclide

Suitable metallic radionuclides (i.e., metallic radioisotopes ormetallic paramagnetic ions) include Antimony-124, Antimony-125,Arsenic-74, Barium-103, Barium-140, Beryllium-7, Bismuth-206,Bismuth-207, Cadmium-109, Cadmium-115m, Calcium-45, Cerium-139,Cerium-141, Cerium-144, Cesium-137, Chromium-51, Cobalt-55, Cobalt-56,Cobalt-57, Cobalt-58, Cobalt-60, Cobalt-64, Copper-67, Erbium-169,Europium-152, Gallium-64, Gallium-68, Gadolinium-153, Gadolinium-157Gold-195, Gold-199, Hafnium-175, Hafnium-175-181, Holmium-166,Indium-110, Indium-i11, Iridium-192, Iron-55, Iron-59, Krypton-85,Lead-210, Manganese-54, Mercury-197, Mercury-203, Molybdenum-99,Neodymium-147, Neptunium-237, Nickel-63, Niobium-95, Osmium-185+191,Palladium-103, Platinum-195m, Praseodymium-143, Promethium-147,Protactinium-233, Radium-226, Rhenium-186, Rhenium-188, Rubidium-86,Ruthenium-103, Ruthenium-106, Scandium-44, Scandium-46, Selenium-75,Silver-110m, Silver-111, Sodium-22, Strontium-85, Strontium-89,Strontium-90, Sulfur-35, Tantalum-182, Technetium-99m, Tellurium-125,Tellurium-132, Thallium-204, Thorium-228, Thorium-232, Thallium-170,Tin-113, Tin-114, Tin-117m, Titanium-44, Tungsten-185, Vanadium-48,Vanadium-49-, Ytterium-169, Yttrium-86, Yttrium-88, Yttrium-90,Yttrium-91, Zinc-65, and Zirconium-95.

As used herein, a “detectable radionuclide” is any suitable radionuclide(i.e., radioisotope) capable of detecting cancer or other neoplasticcells in a diagnostic procedure in vivo or in vitro. Suitable detectableradionuclides include metallic radionuclides (i.e., metallicradioisotopes) and non-metallic radionuclides (i.e., non-metallicradioisotopes).

As used herein, a “therapeutic radionuclide” is any suitableradionuclide (i.e., radioisotope) that possesses therapeutic efficacyagainst cancer or other neoplastic cells in vivo or in vitro. Suitabletherapeutic radionuclides include metallic radionuclides (i.e., metallicradioisotopes).

Chelating Groups

Any suitable chelating group can be incorporated into the compounds ofthe invention. Suitable chelating groups include those disclosed in U.S.Pat. No. 5,739,313. Specifically, the chelating group can be NTA, HEDTA,DCTA, RP414, MDP, DOTATOC, CDTA, HYNIC, EDTA, DTPA, TETA, DOTA, DOTMP,DCTA, 15N4, 9N3, 12N3, or MAG3 (or another suitable polyamino acidchelator), which are described herein below, or a phosphonate chelator(e.g. EDMT). More specifically, the chelating group can be DTPA.

DTPA is diethylenetriaminepentaacetic acid; TETA is1,4,8,11-tetraazacyclotetra-decane-N,N′,N″,N′″-tetraacetic acid; DOTA is1,4,7,10-tetraazacyclododecane-N,N′,N′,N′″-tetraacetic acid; 15N4 is1,4,8,12-tetraazacyclopentadecane-N,N′,N″,N′″-tetraacetic acid; 9N3 is1,4,7-triazacyclononane-N,N′,N″-triacetic acid; 12N3 is1,5,9-triazacyclododecane-N,N′,N″-triacetic acid; MAG3 is(N—[N—[N—[(benzoylthio)acetyl]-glycyl]glycyl]glycine); and DCTA is acyclohexane-based metal chelator of the formula 2

wherein R³ may by (C₁-C₄)alkyl or CH₂CO₂—, which may be attached throughpositions 4 or 5, or through the group R³ and which carries from 1 to 4detectable metal or nonmetal cations (M), monovalent cations, or thealkaline earth metals. Thus, with metals of oxidation state +1, eachindividual cyclohexane-based molecule may carry up to 4 metal cations(where both R³ groups are CH₂COOM). As is more likely, with higheroxidation states, the number of metals will decrease to 2 or even 1 percyclohexane skeleton. This formula is not intended to limit the moleculeto any specific stereochemistry.

NTA, HEDTA, and DCTA are disclosed in Poster Sessions, Proceedings ofthe 46th Annual Meeting, J. Nuc. Med., p. 316, No. 1386. RP414 isdisclosed in Scientific Papers, Proceedings of the 46th Annual Meeting,J. Nuc. Med., p. 123, No. 499. MDP is disclosed in Scientific Papers,Proceedings of the 46th Annual Meeting, J. Nuc. Med., p. 102, No. 413.DOTATOC is disclosed in Scientific Papers, Proceedings of the 46thAnnual Meeting, J. Nuc. Med., p. 102, No. 414 and Scientific Papers,Proceedings of the 46th Annual Meeting, J. Nuc. Med., p. 103, No. 415.CDTA is disclosed in Poster Sessions, Proceedings of the 46th AnnualMeeting, J. Nuc. Med., p. 318, No. 1396. HYNIC is disclosed in PosterSessions, Proceedings of the 46th Annual Meeting, J. Nuc. Med., p. 319,No. 1398.

Bifunctional chelators (i.e., chelating groups) based on macrocyclicligands in which conjugation is via an activated arm attached to thecarbon backbone of the ligand can also be employed as a chelating group,as described by M. Moi et al., J. Amer. Chem. Soc., 49, 2639 (1989)(2-p-nitrobenzyl-1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraaceticacid); S. V. Deshpande et al., J. Nucl. Med., 31, 473 (1990); G. Kuseret al., Bioconj. Chem., 1, 345 (1990); C. J. Broan et al., J. C. S.Chem. Comm., 23, 1739 (1990); and C. J. Anderson et al., J. Nucl. Med.36, 850 (1995)(6-bromoacetamido-benzyl-1,4,8,11-tetraazacyclotetadecane-N,N′,N″,N′″-tetraaceticacid (BAT)).

In addition, the diagnostic chelator or therapeutic chelating groups canbe any of the chelating groups disclosed in Scientific Papers,Proceedings of the 46th Annual Meeting, J. Nuc. Med., Wednesday, Jun. 9,1999, p. 124, No. 500.

A “detectable chelating group” is a chelating group comprising ametallic radionuclide (e.g., a metallic radioisotope) capable ofdetecting cancer or other neoplastic cells in vivo or in vitro.

Specifically, the chelating group can be any one of the carbonylcomplexes disclosed in Waibel et al., Nature Biotechnology, 897-901,Vol. 17, September 1999; or Sattelberger et al., Nature Biotechnology,849-850, Vol. 17, September 1999.

Specifically, the detectable chelating group can be any of the carbonylcomplexes disclosed in Waibel et al, Nature Biotechnology, 897-901, Vol.17, September 1999; or Sattelberger et al., Nature Biotechnology,849-850, Vol. 17, September 1999, further comprising a metallicradionuclide. More specifically, the detectable chelating group can beany of the carbonyl complexes disclosed in Waibel et al., NatureBiotechnology, 897-901, Vol. 17, September 1999; or Sattelberger et al.,Nature Biotechnology, 849-850, Vol. 17, September 1999, furthercomprising Technetium-99m.

As used herein, a “therapeutic chelating group” is a chelating groupcomprising a metallic radionuclide (e.g., a metallic radioisotope) thatpossesses therapeutic efficacy against cancer or other neoplastic cellsin vivo or in vitro. Any suitable chelating group can be employed.

Specifically, the therapeutic chelating group can be any of the carbonylcomplexes disclosed in Waibel et al, Nature Biotechnology, 897-901, Vol.17, September 1999; or Sattelberger et al., Nature Biotechnology,849-850, Vol. 17, September 1999, further comprising a metallicradionuclide. More specifically, the therapeutic chelating group can beany of the carbonyl complexes disclosed in Waibel et al., NatureBiotechnology, 897-901, Vol. 17, September 1999; or Sattelberger et al,Nature Biotechnology, 849-850, Vol. 17, September 1999, furthercomprising Rhenium-186 or Rhenium-188.

Tumors treatable with the compounds and methods of the invention can belocated in any part of the mammal. Specifically, the tumor can belocated in the breast, lung, thyroid, lymph node, genitourinary system(e.g., kidney, ureter, bladder, ovary, teste, or prostate),musculoskeletal system (e.g., bones, skeletal muscle, or bone marrow),gastrointestinal tract (e.g., stomach, esophagus, small bowel, colon,rectum, pancreas, liver, or smooth muscle), central or peripheralnervous system (e.g., brain, spinal cord, or nerves), head and necktumors (e.g., ears, eyes, nasopharynx, oropharynx, or salivary glands),or the heart.

The compounds disclosed herein can be prepared using procedures similarto those described in U.S. Pat. No. 5,739,313, or using proceduressimilar to those described herein. The residue of a molecule comprisingB-10 can be linked to the residue of a compound of formula I asdescribed hereinabove. Additional intermediates and synthetic proceduresuseful for preparing compounds of the invention are disclosed, forexample, in Hogenkamp, H. et al., Synthesis and Characterization ofnido-Carborane-Cobalamin Conjugates, Nucl. Med. & Biol., 2000, 27,89-92; Collins, D., et al., Tumor Imaging Via Indium 111-LabeledDTPA-Adenosylcobalamin, Mayo Clinic Proc., 1999, 74:687-691; U.S.application Ser. No. 60/129,733 filed 16 Apr. 1999; U.S. applicationSer. No. 60/159,874 filed 15 Oct. 1999; U.S. application Ser. No.60/159,753 filed 15 Oct. 1999; U.S. application Ser. No. 60/159,873filed 15 Oct. 1999; and references cited therein.

In cases where compounds are sufficiently basic or acidic to form stablenontoxic acid or base salts, administration of the compounds as saltsmay be appropriate. Examples of pharmaceutically acceptable salts areorganic acid addition salts formed with acids which form a physiologicalacceptable anion, for example, tosylate, methanesulfonate, acetate,citrate, malonate, tartarate, succinate, benzoate, ascorbate,α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts mayalso be formed, including hydrochloride, sulfate, nitrate, bicarbonate,and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made.

The compounds of the present invention can be formulated aspharmaceutical compositions and administered to a mammalian host, suchas a human patient in a variety of forms adapted to the chosen route ofadministration, i.e., orally or parenterally (e.g., by intravenous,intramuscular, intraperitoneal). Preferably, the compounds areadministered parenterally.

The active compound may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form should be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

For illustration, suitable doses of a compound of the invention for usein therapy, in conjunction with neutron capture, include doses in therange of from about 0.1 μg to about 100 μg, e.g., from about 0.5 μg toabout 50 μg, or from about 0.5 μg to 15 μg per treatment. Suitable dosesfor use in imaging or for use in imaging and therapy include doses inthe range of from about 0.1 mg to about 50 g, e.g., from about 0.5 mg toabout 10 g, or from about 0.5 g to 2 g per treatment.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations.

The compounds are preferably dissolved or dispersed in a nontoxic liquidvehicle, such as physiological saline or a similar aqueous vehicle, tothe desired concentration. A preselected therapeutic unit dose is thenadministered to the test animal or human patient, by oral administrationor ingestion or by parenteral administration, as by intravenous orintraperitoneal infusion or injection, to attain the desired in vivoconcentration. Doses useful for treating tumors can be derived, fromthose found to be effective to treat tumors in humans in vitro or inanimal models, such as those described herein below, or from dosages ofother labeled vitamin B₁₂ molecules, previously employed in animaltherapy.

FIG. 2 and FIG. 3 each illustrate the four step synthesis used toprepare three cyanocobalamin-nido-carborane conjugates. o-Carboranecarboxylic acid (2) was prepared by reacting o-carborane withn-butyllithium and carbon dioxide in ether for approximately one hour at−78° C. Treatment with thionyl chloride gave o-carborane carboxylic acidchloride (3), which was allowed to react with 1,4-butanediamine inpyridine to give the amide linked nido-carboranoyl(4-aminobutyl)amide(4). Compound (4) was linked to the b-monocarboxylic acid ofcyanocobalamin, the d-monocarboxylic acid of cyanocobalamin, or both theb- and d-dicarboxylic acid of cyanocobalamin. In each reaction,hydroxybenzotriazole and 1-ethyl-3-(3-dimethylaminoprop-yl) carbodiimidewas added to facilitate the formation of the amide bond.

The invention may be further illustrated by the following examples.

EXAMPLES

Ultraviolet-visible spectra were recorded with a diode arrayspectrophotometer. ¹¹B NMR spectra at 192.656 MHZ with ¹H decouplingwere recorded on Varian 1 NOVA 600 MHZ spectrometer with an 8 mm broadband probe. Approximately 10 mg of the cobalamin-conjugate weredissolved in 1 mL pyridine d₆ in a 5 mm NMR tube. Five hundred scanswere collected with an acquisition time of 0.133 seconds and arelaxation delay of 1 second. Chemical shifts are given relative toBF₃:(CH₃CH₂)₂O.

Mass spectral data was obtained on a Sciex API 365 LC-MS/MS system(Toronto, Canada). Separations were done on a Shimadzu HPLC systemconsisting of two LC-10AD pumps and a SCL-10Avp controller (ShimadzuScientific Instruments, Columbia, Md.). Analytes were monitored by UV at214 NM with an ABI 785 detector. HPLC separations were achieved using aBDS-Hypersil C8 column (150×4.6 mm; 120 A, Keystone Scientific, Inc.,Bellefonte, Pa.). The mobile phase consisted of H₂O:methanol (98:2 v:v)in pump A and H₂O:methanol (2:92 v:v) in pump B. A linear gradient wasused from 5% B to 30% B over 10 minutes and was held at 30% B for 20minutes before returning to initial conditions (mono-carboranesynthesis). The di-carborane required a longer gradient using the samemobile phases; 5% B to 65% B over 25 minutes and held at 65% B for 15minutes before returning to initial conditions. The separations weremonitored by UV absorption at 214 NM. The flow was 1.0 ml/minute and wassplit post-column allowing ˜10 μL to flow into the mass spectrometer.

Mass spectral data was collected using electrospray ionization inpositive mode over a mass range of 300 to 2300 AMU at a dwell time of0.3 ms/0.1 AMU. Synthetic samples were prepared at 5 or 10 mg/ml in pumpA mobile phase and an aliquot injected onto the HPLC (1-5 μL). Retentiontimes of the mono- and di-carborane products were determined to be 13.3minutes and 16.2 minutes respectively. Purification of the b and dmono-carborane products was achieved by collecting fractions at theelution times for several injections. The collected fractions werecombined and dried to a powder. A portion of the purified product wasdissolved in methanol:H₂O (1:1) and reanalyzed by HPLC-MS to ascertainthe purity. o-Carborane, butyllithium (1.6 M solution in hexanes) andputrescine were purchased from Aldrich Chemical Company (Milwaukee,Wis.). The water-soluble carbodiimide 1-ethyl-3(3′-dimethylaminopropyl)carbodiimide and 1-hydroxybenzotriazole were from Sigma (St. Louis,Mo.). Thin layer chromatography (TLC) silica gel plates were obtainedfrom Eastman Kodak Company. The cyanocobalamin-b, d, and emonocarboxylic acids and the b, d-dicarboxylic acid were prepared asdescribed before (see U.S. Pat. No. 5,739,313, and D. L. Anton, et al.,J. Am. Chem. Soc., 1980, 102, 2212-2219), and were provided by ProfessorKahl of the Department of Medicinal Chemistry at the University ofCalifornia, San Francisco.

Example 1 Cyanocobalamin-nido-carborane Conjugates (5, FIG. 2)

Separate reaction mixtures containing 1 g (˜0.66 mmol) of theb-monocarboxylic acid, d-monocarboxylic acid, or the b,d-dicarboxylicacid of cyanocobalamin, hydroxybenzotriazole (810 mg, 6.0 mmol),1-ethyl-3(3-dimethylaminopropyl) carbodiimide (11.4 g, 6.0 mmol) and 4(600 mg, 2.0 mmol) in 100 mL of a water-acetone mixture (2:1) wereadjusted to pH 6.9 with 1N NaOH. The reactions were stirred at roomtemperature and their progress monitored by TLC. After 3 hours, themixtures were concentrated to remove acetone and the resulting aqueoussuspensions were extracted into 92% aqueous phenol. The phenol phaseswere washed with water to remove the water-soluble reactants. One volumeof acetone and three volumes of ether were added to each of the phenolphases and the desired cyanocobalamin-nido-carborane conjugates wereback extracted into water. The aqueous layers were extracted three timeswith ether to remove residual phenol and unreacted (4). Finally theaqueous solutions were evaporated to dryness. The residuals weretriturated with acetone and the desired conjugates isolated asorange-red powders (yields 90-95% based on the cyanocobalamin-carboxylicacids).

The isotope ratios of the final products were also compared totheoretical as follows: Mono product theoretical M+H+(C₇₀H₁₀₉CoB₉O₁₅PN₁₅)—1584.8 (12%), 1585.8 (37%), 1586.8 (74%), 1587.8(100%), 1588.8 (84%), 1589.8 (44%), 1590.8 (16%), (4%). SynthesisI—1584.8 (14%), 1585.8 (37%), 1586.8 (74%), 1587.8 (100%), 1588.8 (88%),1589.8 (48%), 1590.8 (19%), 1591.8 (6%). Synthesis II—1584.8 (15%),1585.8 (40%), 1586.8 (78%), 1587.8 (100%), 1588.8 (86%), 1589.8 (44%),1590.8 (17%), 1591.8 (7%).

Di product theoretical M+H⁺—(C₇₇H₁₂₉CoB₁₈O₁₆PN₁₆)—1815.1 (9%), 1816.1(23%), 1817.1 (47%), 1818.1 (77%), 1819.1 (99%), 1820.1 (100%), 1821.1(77%), (44%), 1823.1 (19%), 1824.1 (6%). Observed synthesis—1815.1(15%), 1816.1 (27%), 1817.1 (51%), 1818.1 (79%), 1819.1 (100%), 1820.1(100%), 1821.1 (78%), 1822.1 (49%), 1823.1 (25%), 1824.1 (12%).

Results included the identification of the b and d carboranecyanocobalamin (CCC) analogs by LC-MS. Products identified weresubsequently separated and purified by HPLC. The purified products werereanalyzed by LC-MS with no starting material detected. MS/MS data wasalso obtained on the b and d CCC analogs to provide further structuralcharacterization. These purified products were then tested in thebiological assays. The e-CCC analog and the di-CCC analog were alsoseparated and identified by LC-MS, however, the preparations containedmore reaction side products resulting in difficult purification. Theisotope ratios of the b- and d-CCC analogs, as well as the di-CCCanalog, were compared to theoretical isotope ratios to provide furtherinformation for identification.

Mass spectra confirmed the presence of eachcyanocobalamin-nido-carborane conjugate. Mass spectroscopy analysis aswell as ¹¹B NMR showed that during the conversion of 3 to 4 (FIGS. 2 and3) the o-carborane nucleus lost a boron atom to yield the nido-carboranederivative 4 (FIG. 3). UV-visible spectroscopy of the final productsshowed maxima typical of cyanocobalamin, indicating that the corrinnucleus was intact and that the 5,6-dimethyl benzamidazole nucleotidewas still attached to the cobalt atom.

The starting material 4 was prepared as follows (see FIG. 2):

a. o-Carborane carboxylic acid (2). A solution of o-carborane (1) (5.0g, 34.7 mmol) in 500 ml dry ether in a 1 L round bottom flask was cooledto −78° C. in a dry ice-acetone bath. The solution was flushed withargon and the flask sealed with a serum stopper. n-Butyllithium (24 mL,1.6 M in hexanes) was slowly injected via a syringe over a period ofabout 20 minutes and the reaction was stirred for an additional 30minutes. Crushed dry ice (10-15 g) was then added and the mixturestirred for 1 hour. The dry ice-acetone bath was removed and thereaction allowed to come to room temperature. The ether and hexanes wereremoved on a rotary evaporator, water (150 ml) was added and unreactedo-carborane was removed by extraction with hexanes (2×100 ml). Theaqueous phase was acidified with concentrated HCl and the desiredproduct extracted with hexanes (4×100 ml). The combined extracts weredried over Na₂SO₄ and evaporated to dryness to give 5.8 g (88.7%) ofo-carborane carboxylic acid (2), which was used without furtherpurification.

b. o-Carborane carboxylic acid chloride (3). o-Carborane carboxylic acid(2.0 g, 10.6 mmol), dried over P₂O₁₄, was dissolved in 30 mlthionylchloride and heated under reflux for 3 hours. The solution wascooled to room temperature, evaporated to dryness and dried over P₂O₁₄(2.05 g, 9.9 mmol, 93%) to provide (3), which was used without furtherpurification.

c. Nido-Carboranoyl(4-aminobutyl) amide (4). The acid chloride (3) wasdissolved in 15 ml dry pyridine and 1,4-diaminobutane (1.12 g, 12.7mmol) was added. The reaction mixture was heated under reflux for 3hours, cooled to room temperature and concentrated. Water (10 ml) wasadded and the suspension acidified with 3 M HCl. The desired product wasextracted with ethyl acetate (4×50 ml), the combined organic layers werewashed once with water, dried over Na₂SO₄ and evaporated to dryness toyield 2.35 g (8.0 mmol, 75%) of (4). Thus far the product has resistedcrystallization from a variety of solvent mixtures, however TLC onsilica gel plates (2-propanol-NH₄OH—H₂O; 7:1:2) showed only oneninhydrin positive compound distinct from the diamine.

Example 2 6-Carboranoylamidopropyl Cobalamin

6-(3-aminoprop-1-yl)cobalamin (300 mg), hydroxybenzotriazole (270 mg),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (382 mg) and o-carboranemonocarboxylic acid were dissolved in water (50 ml) and acetone (20 ml)and allowed to react at room temperature for 3 hours. The reactionmixture was concentrated to remove acetone and desalted via phenolextraction. The concentrated aqueous solution was crystallized fromaqueous acetone to give the title compound.

The intermediate 6-(3-aminoprop-1-yl)cobalamin was prepared as follows:Cyano cobalamin (500 mg) was dissolved in 100 ml deoxygenated watercontaining 10 mg CoCl₂, and reduced with sodium borohydride to give thecorresponding Co (I) compound. After 30 minutes, 3-chloropropyl aminehydrochloride (130 mg) dissolved in 5 ml deoxygenated ethanol was added.After one hour, at room temperature, the mixture was desalted via phenolextraction. The aminopropylcarbolamine was back extracted into waterafter the addition of 1 volume of acetone and 3 volumes of ether. Theaqueous solution was concentrated and the desired6-(3-aminoprop-1-yl)cobalamin crystallized from aqueous acetone (yield510 mg).

Example 3 DTPA-aminopropylcarbalamin (DAPC)

Using a coupling procedure similar to that described in Example2,6-(3-aminoprop-1-yl)-cobalamin and DTPA were coupled in the presenceof hydroxybenzotriazole to give the title compound.

Example 4

In vitro biological activity of the carborane cyanocobalamin analogs. Toassess the in vitro binding of the carborane cyanocobalamin (Example 1,CCC) and DTPA-aminopropylcobalamin (Example 3, DAPC) analogs to thetranscobalamin proteins, the unsaturated Vitamin B12 binding capacity(UBBC) assay (see D. A. Collins and H. P. C. Hogenkamp, J. NuclearMedicine, 1997, 38, 717-723) was performed. Serum was obtained from 5patients being evaluated for pernicious anemia at the Mayo Clinic. Thepatients' serum first underwent a routine clinical UBBC as previouslydescribed. To determine if the CCC and DAPC analogs would inhibit Co-57cyanocobalamin from binding to the transcobalamin proteins, the excessserum from the 5 patients underwent modified UBBC.

Specifically, under dim light, 0.4 ml serum was treated with 4 μL(concentration 10 μg CCC per ml normal saline) of the 2 CCC analogs,carborane-d-cyano-cobalamin and carborane-b-cyanocobalamin, and DAPC.The analogs were incubated for 20 minutes at room temperature with thepatient's serum. Both the clinical run and the analog treated sampleswere assayed for UBBC as usual.

The analogs competitively blocked Co-57-cyanocobalamin from binding tothe transcobalamin proteins. Therefore, the cpm of the modified UBBCassay was significantly lower than that of the clinical runs. Thepercent PB of the analogs to transcobalamin protein was calculated asfollows (PB=100−CCC_(UBBC) cpm/clinical UBBC cpm×100). The averagepercent binding (PB) of the 5 solutions (n=10 for each solution, i.e.,two modified UBBC assays per patient) for the carborane-d cyanocobalaminand carborane-b cyanocobalamin was 35.75% and 92.93% respectively, and98.21% for DAPC.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

1. A compound of formula I:

linked to at least two molecules comprising B-10, wherein X is CN, OH,CH₃, adenosyl, or a molecule comprising B-10; or a pharmaceuticallyacceptable salt thereof.
 2. The compound of claim 1, wherein at leastone of the molecules comprising B-10 is directly linked to the6-position of the compound of formula I or is directly linked to the b-,d-, or e-carboxamide of the compound of formula I.
 3. The compound ofclaim 1, wherein at least one of the molecules comprising B-10 is linkedby a linker to the 6-position of the compound of formula I or is linkedby a linker to the b-, d-, or e-carboxamide of the compound of formulaI.
 4. The compound of claim 1, wherein the molecule comprising B-10contains I to about 20 boron atoms, inclusive.
 5. The compound of claim1, wherein the molecule comprising B-10 is an amino acid, acarbohydrate, a nucleoside, or a carborane.
 6. The compound of claim 1,wherein the molecule comprising B-10 is o-carborane, m-carborane, orp-carborane.
 7. The compound of claim 1, wherein the molecule comprisingB-10 is o-carborane.
 8. The compound of claim 3, wherein at least onelinker is of the formula W-A-Q wherein A is (C₁-C₆)alkyl,(C₂-C₆)alkenyl, (C₂-C₆)alkynyl, (C₃-C₈)cycloalkyl, or (C₆-C₁₀)aryl,wherein W and Q are each independently —N(R)C(═O)—, —C(═O)N(R)—,—OC(═O)—, —C(═O)O—, —O—, —S—, —S(O)—, —S(O)₂—, —N(R)—, —C(═O)—, or adirect bond; wherein each R is independently H or (C₁-C₆)alkyl.
 9. Thecompound of claim 8, wherein W is NH₂ or COOH and Q is NH₂ or COOH. 10.The compound of claim 8, wherein A is (C₁-C₆)alkyl.
 11. The compound ofclaim 3, wherein at least one linker comprises a therapeuticradionuclide or a diagnostic radionuclide.
 12. The compound of claim 11,wherein the therapeutic radionuclide is a metallic radionuclide.
 13. Thecompound of claim 11, wherein the diagnostic radionuclide is a metallicradionuclide.
 14. The compound of claim 11, wherein the diagnosticradionuclide is a non-metallic radionuclide.
 15. The compound of claim3, wherein at least one linker is poly-L-glutamic acid, poly-L-asparticacid, poly-L-histidine, poly-L-ornithine, poly-L-serine,poly-L-threonine, poly-L-tyrosine, poly-L-leucine,poly-L-lysine-L-phenylalanine, poly-L-lysine orpoly-L-lysine-L-tyrosine.
 16. The compound of claim 1, wherein thecompound of formula I is also linked to a linker comprising a detectableradionuclide or a therapeutic radionuclide.
 17. A composition comprisinga compound of claim 1 and a pharmaceutically acceptable carrier.
 18. Amethod of treating a tumor in a mammal comprising: a) administering tothe mammal an effective amount of a compound of formula I:

 linked to at least two molecules comprising B-10, wherein X is CN, OH,CH₃, adenosyl, or a molecule comprising B-10; or a pharmaceuticallyacceptable salt thereof, in combination with a pharmaceuticallyacceptable vehicle; and b) administering neutron capture therapycomprising subjecting said mammal to thermal neutron irradiation at thesite of said tumor for a time and under conditions effective to treatsaid tumor.
 19. A method of imaging a tumor in a mammal comprising: a)administering to the mammal an effective amount of a compound of formulaI:

 linked to at least two molecules comprising B-10, wherein X is CN, OH,CH₃, adenosyl, or a molecule comprising B-10; or a pharmaceuticallyacceptable salt thereof, in combination with a pharmaceuticallyacceptable vehicle; and b) detecting the presence of the compound. 20.The method of claim 19, further comprising treating the tumor withneutron capture therapy.